BIOCELL ISSN 0327 - 9545 2012, 36(2): 57-62 PRINTED IN

Seasonal testicular changes in minutus Peters, 1872 (Anura, )

ADELINA FERREIRA1,* AND MAHMOUD MEHANNA2

1. Universidade Federal de Mato Grosso (UFMT), Instituto de Biociências, Cuiabá, MT, Brasil. 2. Universidade Estadual Paulista Julio de Mesquita Filho (UNESP), Laboratório de Biologia de Peixes, Pós-graduação em Zoologia, Botucatu, SP, Brasil.

Key words: spermatogenesis, reproduction, anura, ultrastructure, Chapada dos Guimarães.

ABSTRACT: The reproductive cycle in anurans may be either continuous or discontinuous. These differ- ences may be connected to seasonal climate changes and/or to anthropic activity. Forty adult male individuals of the Dendropsophus minutus species were collected during one year, in the municipality of Chapada dos Guimarães (Mato Grosso, ). The testicles were studied under light and transmission electron micros- copy. No variations were observed when the diameter of the seminiferous tubules and the thickness of the interstitial tissue were studied. However, changes in spermatogenesis were conspicuous and indicated that the reproductive cycle of D. minutus in Chapada dos Guimarães is discontinuous and seems related to variations in air temperature and rainfall.

Introduction Trinidad and, moving south, through , and Brazil as far as , and Argentina (Ribeiro Breeding activity of most anurans is associated with et al., 2005). the rainy season both in the temperate zone (Wiest, 1972; Chapada dos Guimarães comprises a wide plateau Salvador and Carrascal, 1990) and the tropics (Heyer, with an altitude varying between 600 and 850 meters. 1973; Barbault and Rodrigues, 1978; Hoogmoed and In Mato Grosso, where the most elevated areas are Gorzula, 1979; Toft and Duellman, 1979; Aichinger, formed by plateaus, the altitude associated with venti- 1987; Gascon, 1991; Duellman, 1995). Yet there are no lation constitutes the geographical factor that most in- descriptions of anuran reproductive activity in the Bra- fluences the variations in temperature and rainfall zilian Cerrado (savanna) bioma. (Maitelli, 2005). Chapada dos Guimarães is located in Dendropsophus minutus (Hylidae) is a small size the center-south of the state of Mato Grosso and is a anuran measuring 20-25 mm (crown-rump length). Its watershed between the Platina and Amazon basins geographical distribution ranges from the lowlands up (Maitelli, 2005). During the year there are two notably to 2000 meters in altitude, from low areas to the east of different seasons, with regard to pluviometric precipi- the Andes in , passing through and tations: the rainy season (spring and summer) and the dry season (autumn and winter). This seasonal varia- tion may be characterized by six rainy months and six *Address correspondence to: Adelina Ferreira. Universidade Federal de Mato Grosso (UFMT), Instituto de hot months, with oscillations between extreme heat and Biociências Cuiabá, MT, BRASIL. dry cold (Fig. 3). E-mail: [email protected] The anuran testicles show a seminiferous epithe- Received: April 12, 2011. Revised version received: July 14, 2012. Accepted: July 17, 2012. lium with cysts formed from the association of germ 58 ADELINA FERREIRA and MAHMOUD MEHANNA cells with Sertoli cells, and all cells present in the same Grosso, Brazil. This area belongs to the biogeographical cyst are at the same stage of differentiation (Ferreira et domain of the Cerrado (Maitelli, 2005). The average al. 2008; 2009). monthly averages of temperature and rainfall during 2006 (Fig. 3), were supplied by INMET (Instituto Nacional de Metereologia - 9th District of the municipality of Cuiabá, Material and Methods Mato Grosso State).

Study area Light microscopy

Animals were collected in the permanent Monjolinho Adult male individuals were collected monthly from stream on the Buriti farm (15°36’S; 56°03’W) in the July 2004 to June 2005 (N=50). They were measured municipality of Chapada dos Guimarães, state of Mato and fixed in 10 per cent formalin, preserved in 70 per

FIGURE 1: Spermatogenesis stages of Dendropsophus minutus. a-c. Light microscopy. Bar is 20μm. a. Primary spermatogonia. b. Primary spermatocyte. c. Secondary spermatocyte. d-g. Transmission electron microscopy. d. Primary spermatocyte. Bar is 580 nm. e. Secondary sper- matocyte. Bar is 580 nm. f. Early spermatid, the arrow indicates the pro-acrosomal vesicle. Bar is 750 nm. g. Early spermatid in phase of compacting chromatin. Bar is 580 nm. h. Light Microscopy, late spermatids with nuclear elongate and compacting chromatin. Bar is 20 μm. i-m. Transmission electron microscopy. i. Late spermatid, in phase compacting chromatin and still evident nucleolus (n), formation of nuclear fossa is the implantation of the midpiece with centriole (c) and showing initial dense bodies (p). Bar is 1 μm. j. Late spermatid with more accented cromatin compactation and implantation nuclear fossa of the midpiece, with central centriole (c). Bar is 580 nm. k. Acrosome apical, nucleus (n), subacrosomal cone (sc), nuclear space (ns) and acrosomal vesicle (av). Bar is 430 nm. l. Middle piece, axoneme (ax), fibrous sheath (fs), mitochondrial ring (mr). Bar is 310 nm. m. End piece, axoneme (ax) and plasma membrane (m). Bar is 310 nm. REPRODUCTIVE CYCLE OF Dendropsophus minutus 59 cent alcohol and deposited in the Zoological Collection Transmission electron microscopy of Vertebrates of the Mato Grosso Federal University. One testicle was obtained from each , and was Testes were fixed overnight at 4ºC in a 0.1M so- fixed in 5% paraformaldehyde, dehydrated in increas- dium cacodylate buffer solution (pH = 7.2) containing ing graduations of ethanol, infiltrated and embedded in 2.5% glutaraldehyde. After, they were fixed following a methacrylate glycol resin; 3μm sections were obtained the above protocol, they were post-fixed for 2 h in 0.1M and stained with 1% toluidine blue. sodium cacodylate buffer solution (pH = 7.2) contain-

FIGURE 2. a1. Histological images showing the general aspect of the seminiferous tubule of Dendropsophus minutus through- out the year. a. January. b. February. c. March. d. April. e. May. f. June. g. July. h. August. i. September. j. October. k. November. l. December. Bar is 20μm. 60 ADELINA FERREIRA and MAHMOUD MEHANNA ing 1% osmium tetroxide. Next, they were dehydrated matogenesis throughout the year, we observed that there in acetone and embedded in LR White resin. Ultra-thin occurs a subtle, but noticeable difference in the propor- sections were stained with uranyl acetate and lead cit- tion of germ cells. However, no variation was noted in rate and observed with a transmission electron micro- the interstitial tissue. All germ cell stages are found in scope (Zeiss, Leo 906). every month of the year (Fig. 2). However, both elon- gated spermatids and spermatozoa are more frequent than the other cell types from January to April (Figs. 2 Results a-d) and an appreciable reduction in frequency of these elongated cells was observed from May to September, The testis of Dendropsophus minutus are round and when spermatogonia and spermatocytes were more fre- whitish, without noticeable variations in their macro- quently found (Figs. 2 e-i). In October to December a scopical appearance throughout the year. subtle increase in the proportion of elongated cells re- The structural and ultrastructural analysis of germ commenced (Figs. 2 j-l). cells showed that the primary spermatogonia is an iso- The presence of sperm in the lumen of the seminif- lated cell that does not form a cyst and is larger in size erous tubules enabled us to identify the period of repro- than the other cells and is located close to the seminif- ductive activity (November to February) which was co- erous tubule wall. Its cytoplasm possesses a great af- incident with the highest rainfall sperm (Fig. 3). In the finity for staining, enabling good visualization of cell following months there was a tendency for spermato- limits under light microscopy (Fig. 1a). Secondary sper- genesis to decrease and about half of the tubules show matogonia are smaller cells organized in cysts of a dark no sperm in the lumen in August, just before the in- appearance. The primary spermatocyte has a large crease in rainfall (Fig. 3). However, no obvious changes nucleus with loose chromatin and a thread-like appear- in the diameter of the seminiferous tubules or in the ance (Figs. 1 b and d). Secondary spermatocytes are extent of the interstitial tissue were observed in the smaller than the primary ones, with a roundish and course of the year. strongly colored nucleus, the chromatin of which is much more compact (Figs. 1 c and e). Spermatids are found in two stages of morphological differentiation: roundish or early and elongated or late. The early sper- matids have a spherical nucleus, with relatively com- pact chromatin, while the elongated ones have a longi- tudinally extended nucleus (Figs. 1 f and g). The ultrastructure shows the formation of a pre-acrosomal vesicle in one of the nuclear poles of the round early spermatids (Fig. 1f). Following cell differentiation, the nucleus of the late spermatid shows an increasingly more compact chromatin, elongating itself and transforming itself into spermatozoid, which appears strongly colored, and thinner than the spermatid (Figs. 1 h-j). Sperm have FIGURE 3. Average monthly variations in tem- a head composed of a nucleus whose chromatin is highly perature and rainfall during the studied period. compacted, with few electron-lucent spaces, and an acrosome with an acrosomal vesicle (Fig. 1k). The middle piece is implanted in the nucleus through the Discussion penetration of a pair of perpendicular centrioles sur- rounded by groups of electron-dense granules (Figs. 1 i The seminiferous tubules of anurans showing con- and j). This piece is constituted by a mitochondrial col- tinuous cycles, present cysts at various stages of matu- lar (4 to 8 mitochondria) and a typical axoneme (9+2) ration, with sperm production throughout the year, as it with associated dense fibers (Fig. 1l). Between the ax- is frequently found in tropical and subtropical species oneme and the mitochondria there is a ring of dense (Saidapur, 1983). However, germ cells mature uniformly fibers (Fig. 1l). The flagellum is simple, consisting of a in each tubule in anuran species exhibiting discontinu- typical axoneme and the plasma membrane (Fig. 1m). ous cycles. Notwithstanding, males of some anuran spe- Upon analyzing the structural variations in sper- cies of the temperate zone enter a sexually quiescent REPRODUCTIVE CYCLE OF Dendropsophus minutus 61 period after the reproductive period, and spermatogen- with the greatest threat to biological diversity and the esis is interrupted for some months (Lofts et al., 1972). loss of (Primack and Rodrigues, 2001; Balmford All differentiation stages of spermatogenesis are et al., 2002). The Cerrado has for some time suffered present in Dendropsophus minutus in October, reach- high rates of biodiversity loss due to human occupation ing maximum development in February. This is similar and agricultural expansion (Klink and Moreira, 2002). to observations in Hyla pulchella andina (Montero and For this reason, urgent conservation strategies are im- Pisanó, 1992). In April, when the dry season starts, we portant for this bioma. observed that spermatogenesis decreases in compari- The decline of populations, mainly in son with the preceding months, and this tendency con- Australia, Central America and the Pacific north-east tinues until reaching a peak in August, when secondary of the United States, has been the focus of research spermatogonia become the predominant cells. This pat- projects over the last 12 years (Lanoo, 2005) but am- tern of spermatogenesis is very similar to the spermato- phibian populations in the tropics (as in Ecuador and genic cycle of Telmatobius pisanoi (Montero and Pisanó, Brazil) have been insufficiently monitored (Stuart et al., 1990), which halts the spermatogenic process in Au- 2004). The causes of amphibian populations’ decline, gust, when secondary spermatogonia predominate in the both within and outside protected areas, are still uncer- seminiferous tubules. A reactivation of spermatogen- tain, but a consensus exists that four main factors may esis is indicated by an increase in spermatocyte number be involved: climate change, pollution, increase in ul- in September. traviolet B (UVB) radiation and infectious diseases Up to now, the spermatogenic process has received (Lanoo, 2005). These factors render the group wanting little attention in anurans. The initial phases have only in terms of swift action for conservation strategies. been described in Xenopus laevis (Pipidae) (Kalt, 1976). Similar findings are reported here for D. minutus, mainly in relation to spermatogonia and spermatocyte morphol- Acknowledgements ogy, and we have added a few more cytoplasmatic char- acteristics. The research was supported by the FAPEMAT Among vertebrates, present an enor- (Fundação de Amparo a Pesquisa do Estado de Mato mous diversity in their reproductive strategies (Duellman Grosso, process # 0769/2006). and Trueb, 1994), and this is reflected as large differen- tiation and variability of the caudal filament, including the protean appendages and membranous parts that this References region (Taboga and Dolder, 1993). The caudal filament presents a typical axoneme Aichinger M (1987). Annual activity patterns of anurans in a sea- sonal Neotropical environment. Oecologia 71: 583-592. (9+2) in D. minutus. Similar axonemes without any cau- Asa CS, Phillips DM (1988). Nuclear shaping in spermatids of the dal annex have been observed in R. pipiens and R. Thai leaf Megophrys montana. Anatomical Record 220: clamitans (Poirier and Spink, 1971) and some 287-290. 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